The party's state leader, Steve Dickson, on Saturday claimed primary school students were being taught how to masturbate under the program.
"We are having little kids in grade four at school, young girls being taught by teachers how to masturbate, how to strap on dildos, how to do this sort of stuff -- that is the real problem in this country."
But an angry premier said the Safe Schools program was not taught in Queensland classrooms and Mr Dickson's claims were wrong.
"I'm lost for words, I'm actually lost for words that someone would dare say such things that are simply and utterly not true."
Deputy Premier Jackie Trad said One Nation was telling lies.
"I have got a child in Year 4 and I can guarantee they are not being taught the ridiculous, loopy things Steve Dickson talked about yesterday," Ms Trad said on Sunday.
